1. Field of the Invention
This invention relates to a process for preparing a thermally stable, readily transportable, lyophilized enzyme, terminal deoxynucleotidyl transferase (TDT), and the freeze-dried enzyme resulting therefrom.
2. Description of the Prior Art
An enzyme is a protein with catalytic properties due to its power of specific activation. The characteristic property of enzymes is their power of catalyzing certain definite chemical reactions in aqueous media. To study enzymes, as well as to use them in many applications, it is necessary to remove them from their natural environment and isolate them for use or storage. Under unsuitable conditions, these materials undergo conformational changes, denaturation and inactivation. In removing them from their natural environment, and in their subsequent handling, the first consideration must always be to avoid inactivation.
Success in dealing with enzymes depends upon avoiding conditions under which they are unstable, and, while conditions vary with the particular enzyme in question, in general, high temperature environments are especially to be avoided with all enzyme materials, with many enzyme treatments carried out at or near 0.degree. C. as a matter of course.
In most cases, there is inactivation of enzymes on standing, even under the most favorable conditions, and the storing of enzymes without loss of activity is a significant problem. When an enzyme solution can be frozen and thawed without loss of activity, it is sometimes most convenient to keep it in the frozen state at a temperature of about -20.degree. C., in which state it may be stored stably for from a few weeks to several months. However, this method entails special handling and equipment, and the transportation of these frozen enzymes creates similar problems in handling and equipment. Drying of enzymes is another method which has been used, but problems occur here also, and the shelf-life stability of the dried enzyme is not uniform.
Terminal deoxynucleotidyl transferase (TDT) was first discovered over twenty years ago and purified to homogeneity from animal thymus glands in 1971. Like other DNA polymerases, it forms or extends the length of polydeoxynucleotide, requires low concentrations of magnesium or other divalent metal ions as cofactor, and utilizes the deoxynucleotide-5'-triphosphates as substrate. The unique feature of TDT is that, unlike other DNA polymerases, it does not require a template, i.e., a first strand of DNA to be copied in forming the classical double-stranded helix. Rather, TDT extends a "primer," which must be at least three deoxynucleotide residues in length, using whatever deoxynucleotide-5'-triphosphate is available. The product formed is typically single-stranded DNA. TDT is now widely used in genetic engineering and in the synthesis of certain unique types of DNA polymers.
TDT has been commercially available since about 1971. The commercially available form of TDT has always been a buffered, aqueous 50% glycerol solution of the highly purified enzyme, the glycerol acting as a stabilizing agent and bacteriostat for the enzyme. Other methods of stabilizing the enzyme, including freezing and freeze-drying, have reportedly been unsuccessful.
The increase in the use of TDT for various types of biochemical research has resulted in an increase in the need for a storage-stable, easily transportable TDT enzyme product. Unfortunately, prior to the present invention, the commercially available products have suffered from the disadvantage that they possess very poor thermal stability, thus requiring carefully modulated thermal control during shipping and storing in order to maintain a substantial degree of enzymatic activity.
While Methods in Enzymology, Volume XXII, Pages 32-38, 1971 and Neubeck, U.S. Pat. No. 4,180,917, suggest that lyophilization of enzymes requires removal of all ions by dialysis prior to lyophilization, removal of all ions from solutions of TDT results in protein aggregation, precipitation of the enzyme, and subsequent loss of essentially all enzymatic activity.
Therefore, a need has continued to exist for terminal deoxynucleotidyl transferase of high thermal stability, capable of being maintained in storage or shipping at ambient temperatures, which, upon reconstitution, still maintains substantially all of its biocatalytic activity.